I have studied and worked on antivirals for more than a decade and for the first time I am excited to see the whole world put their undivided attention to the filed has been greatly ignored for so long. And for the first time I have to halt my little undergraduate antiviral research since this coronavirus pandemic. You have to admit there is a little sense of irony in it. But we should know, right now the best strategy to seeking for a cure is to repurpose the drugs on the market (such as chloroquine) or look into the drug candidates in the late development stage (such as Remdesivir). We just simply can’t afford to wait on developing a brand-new compound from the scratch. (The average time for developing a new drug is about 10 years). Now for me as an organic/medicinal chemist there is not much to do other than read, write and constantly answer questions from anyone I know happens to remember that I have something to do with viruses. But more I read, the little I know. For those little things I know about CoV-19, I would like to share with you as listed topics. Call it a hunch I think there will be more of you would like to join the research when it is over.
Topic 1: Spike protein, the famous S protein
The key to the door: interaction with receptor proteins
If you have seen any pictures, imagines or art works of CoV-19, you can’t ignore that signature red spikes stick out of the viral surface like little mushrooms (why it is always red in pictures? that I don’t know.) Those are Spike protein or S protein. They are the first part of virus interacts with cell surface protein, namely receptors. Receptors are host cell surface (or membrane) proteins with various cellular functions. For SARS-CoV-1 and SARS-CoV-2 (CoV-19), the receptor is angiotensin-converting enzyme 2 (ACE2), which is abundantly found on lung and small intestine cells. ACE plays a role in generating the hormone angiotensin II. Angiotensin II is a strong vasoconstrictor which results in increased blood pressure. So many drugs on the market for high-blood pressure are ACE inhibitors. For MERS-CoV, another deadly, actually more deadly human coronavirus, the key is dipeptidyl peptidase 4 (DPP4). Those interaction are important for our immune system defense. In other words, the S protein is important antigen for host immune response and towards that direction it is the target for vaccine. The goal is to deliver something highly mimic the S protein successfully into our body and triggers our immune response, best of all to generate neutralizing antibody, the “fake” receptors to hold S protein from attaching to the cells.
The pathway to get inside: fusing into the cytoplasm
Let’s get back to our viral S protein. The function of the S protein is not only to hook up with the cell but also to bring two membranes (viral and cellular) close enough to fuse together and open up a portal for viral RNA entering the cellular plasma. If we exam the S protein gene for each function components we would get two major parts: s1 as the Receptor Binding Domain and s2 as the Membrane Binding Domain, in charging for two major jobs: job one as binging with the receptor and job two as fusing into the membrane. Binding to the receptor happens spontaneously, while fusing into cellular membrane needs some kicks. It is triggered by cleavage on the protein site s2’ and for some CoV (such as MERS-CoV), another one on s1/s2. But this fusion could happen in two different scenarios, which says something in terms of infectivity.
A) Get the troops in by the door (early pathway)
The ultimate mission for viruses is to self-replicate and to achieve that is to get their viral genome (RNA or DNA, in our case of CoV is the positive single strand RNA) expose to and processed by our sophisticated cellular translation machinery. That means viral RNAs or DNAs need to find out a way to get out their protein shells and reach to the ribosome inside the cell. There are two pathways or strategies for coronavirus to reach the goal. The first one is called the early pathway, which means the portal opening for viral RNA (or membrane fusion) is started immediately after viral binging to the receptor. Like what we mentioned before, to trigger the fusion the S protein has to be cut. The sites (s2’) are quite consistence across the SARS-CoV-1, SARS-CoV-2 and MERS-CoV, between the amino acids arginine and Serine (R-S). Then the suitable scissors for the task is a group of enzymes called protease, more specifically serine protease. There are many of studies looking for the relationship between the cleavage induced infusion with viral infections and the choice of proteases. Some of them are absolutely genius. For instance, one study shows when you treat with retroviral pseudoparticles (the viral surrogate expressing SARS-CoV S protein) with trypsin (plasma serine protease, playing a role in digestion) after the receptor binding, it would result the effective infection, but not before. That experiment tells us the timing for the cleavage is absolutely crucial, otherwise the CoVs would be more infectious and infect more cell types, since we have many of those proteases floating outside of cells. The type II transmembrane serine proteases (TTSPs), in particular the transmembrane protease/serine subfamily member 2 or 4 (TMPRSS2 or 4) sitting on the plasma membrane surface (the little blue mouth in the picture) are the best fit to provoke this early fusion pathway. There are some subtle differences between SARS and MERS in utilizing the early pathway, which later started the lab-made CoV-19 conspiracy.
MERS-CoV but not in SARS-CoV-1 S protein contains a cleavage site at S1/S2 could be recognized by furin or furin-like proteases found in the trans-Golgi network (TGN), which is the place for assembling newly formed viral practices in the late stage of viral replication circle. Here is the meaning of this, after the first MERS-CoV enters the cell, it will start the replication cycle, make more viral genome and viral proteins. All of those viral components will be passed to the TGN, the assembling site. At there the newly formed MERS viral S protein will be pre-cut at s1/s2 site. This pre-cleavage will promote the cleavage at S2’ site for those newly formed viral particles during the next round of infection. In other words, those new generation of MERS virus are more accessible to the protease and more efficient for the fusion process. But that doesn’t mean SARS-CoV is losing the battle. Evidence shows that higher binding affinity would make the conformational changes facilitate for S2’ cleavage. Indeed, the SARS-CoV-1 S protein is 10 to 20-fold higher affinity to the receptor compared to MERS S protein, which in a sense compensate benefit from s1/s2 pre-cleavage.
In February, one research paper unexpectedly found in SARS-CoV-2 (CoV-19) S protein also possesses a furin cleavage site at the S1/S2 region, potentially could be cut during the biosynthesis of new viral particles. Since the receptor binding affinity for SARS-CoV-2 S protein is already much higher than that to SARS-CoV-1 S protein, does this new cleavage site mean even more efficient infusion? If you feel this question is not too chilling, here is another one even more sensational. Is this site was inserted by scientists and subsequently a living evidence to prove the CoV-19 is indeed a product of bioengineering? While the first question remains undetermined. But the evidence shows another SARS-CoV, which highly resembles the CoV-19 found in pangolin also possesses the furin cleavage site. That could be the evolutional track for this CoV-19 virus, which muted after it jumped from the bat to pangolin. After all, nature is always the better biologist.
B) A trojan horse (late pathway)
If you think those coronaviruses will lose their way in when there are no membrane-bound proteases, then you will be disappointed. After bind to the receptor, the SARS and s1/s2 un-cleaved MERS coronaviruses could be engulfed by the cell membrane (endocytosis) like a trojan horse. Then the bubble-like endosome membrane would be teared open from inside trigged by low PH value. All for only 30 mins slower than the early pathway. The strategy reminds me another very successful virus, our longtime enemy, influenza. As matter of fact one of the oldest anti-influenza drug amantadine (approved by FDA in 1968) was based on inhibiting the proton pump (M2 ion channel) to prevent the PH dropping. It was later discontinued due to the drug resistance. The low PH may induce the conformational changes of S protein (like the mechanism for influenza) but it could also active a group of the endosomal proteases, cathepsins to cut and start the fusion of S protein. The cutting site for cathepsins is not quite clear or may vary across viruses, in addition, different proteases may also result different cutting sites. The highly flexibility for choosing the scissors or the cutting locations makes them highly infectious.
Before I turn the last page, there is one more question I haven’t touch yet. Why the S protein need to be cut to initiate the infusion. That is such an organic chemistry question. You see, the virus particle is always surrounded by water (the aqueous plasmatic environment). But the fusion peptide (FP) is fat like (lipophilic or
hydrophobic) since cell membrane is constructed by lipids or fats. It is highly disadvantage to expose this fat part to the surrounding too early. Only when two membranes (viral and cellular) are getting close, the S protein will be cut by proteases to expose the fusion peptide. This lipophilic anchor will then insert into the cell membrane to initiate the membrane fusion.